The following description provides a summary of information relevant to the present invention. It is not an admission that any of the information provided herein is prior art to the presently claimed invention, nor that any of the publications specifically or implicitly referenced are prior art to the invention.
In the art of electronically addressable microchips that are used to direct biomaterials, such as nucleic acids and proteins, from one point in a solution to another, the microchips should be designed so that electric potential from the microchip electrodes will translate to the solution overlying the microchip such that any electrochemistry occurring from the electrode surface will neither damage the electrodes themselves, nor any biomaterials in the solution. Generally, protection from such damage is provided by the use of a porous membrane layer deposited over the microchip electrodes. Usually, such layer comprises materials derived from natural or synthetic polymers such as agarose or polyacrylamide, respectively. These types of materials allow electrochemical products generated at the electrode surface to travel through their porous matrix or `permeation layer` and into the solution immediately above the electrodes.
Although materials such as those noted above have been found useful in the role of a porous membrane having desired qualities, it has been found that because of the methodologies commonly used to layer such membranes onto the microchip substrate, the membranes are prone to separate or `delaminate` from the electrode surface. It is believed this delamination is caused by a change in the chemical make-up at the interface between the permeation layer and the electrode resulting from the application of electronic potential at the electrode and by physical disruption from charged ions and gases emanating from the electrode. Such delamination can be viewed from the standpoint of `microdelamination` and `macrodelamination`.
Microdelamination involves the electrochemical degradation of the chemical interface between the permeation layer and the electrode itself. It is observed by the formation of raised bulges in the permeation layer, or by ringlets visible due to defraction of light from the delaminated layer when appropriately viewed by a confocal microscope and results in the loss of consistency in permeation layer performance (possibly due to the loss of control over the electric field uniformity). Macrodelamination, on the other hand, is caused by a mismatch of the surface energies between the permeation layer and the chip substrate and results in permeation layer peeling (lift-off) which can extend across the entire microchip surface. Since the permeation layer provides a means for chemical anchorage of analytes present in the liquid overlay, its physical loss by macrodelamination results in catastrophic chip failure during bioassays.
Electronically addressable systems such as the microchips considered herein follow Ohm's law which establishes the relationship between the voltage drop (V) between two electrodes (i.e., the anode, placed at a positive potential and the other, the cathode, placed at a negative potential), and the electric current (I) which flows between these electrodes, as follows: EQU V=R.times.I (1)
where R is the electrical resistance of the medium between the anode and the cathode. In systems where a permeation layer is present over such electrodes, the value of R is greatly determined by the physical and chemical nature of said permeation layer. Thus, according to formula (1), the difference between the electronic potentials applied to the electrodes is directly proportional to the intensity or density of the electric current which flows through them. The invention described in this Letters Patent uses a relationship between electric current and voltage wherein electric current densities are at least 0.04 nA/.mu.m.sup.2 and/or voltage drops are between 1 and 3 V. The electric current density is defined as the electric current divided by the area of the electrode used to support it.
Additionally, the effectiveness of the translocation of charged biomolecules such as nucleotide oligomers within an electronically-driven system such as that described herein depends on the generation of the proper gradient of positively and negatively charged electrochemical species by the anode and cathode, respectively. For example, effective nucleic acid (i.e. either DNA or RNA) transport may be accomplished by generation of protons and hydroxyl anions when the potential at the anode is greater than +1.29 V with respect to a `saturated calomel electrode` (SCE). When subjected to such demanding operating conditions, noncovalently-attached permeation layers prove to be unsatisfactory since such systems are likely to experience micro- and sometimes macrodelamination. Moreover, the transport efficiency of charged molecules increases with increasing current density, thus driving the desire for operation at higher voltage drops and current densities and, thus, the need for evermore robust permeation layers.
Therefore, a need still remains for methodologies for keeping permeation layers from delaminating from electronic microchip substrates and particularly from the electrode pads themselves. We have discovered an improvement in permeation layer attachment chemistry that provides a significant increase in permeation layer performance. Specifically, we have solved the problem of micro- and macrodelamination by discovery of a covalent chemistry linkage system that, as applied to electronically addressable microchip art, can be incorporated between the microchip and the permeation layer matrix. This chemistry is applicable to a variety of permeation layer compositions, including polymers, hydrogels, glyoxylagarose, polyacrylamide, polymers of methacrylamide, materials made from other synthetic monomers, and porous inorganic oxides created through a sol-gel process, and is able to withstand current densities of at least 0.04 nA/.mu.m.sup.2 and/or voltage drops between 1 and 3 V.